ABSTRACT DNA replication is central to human genome integrity and is intimately tied to large-scale 3D genome architecture and cell lineage specification, yet we still do not have reliable maps of replicon organization nor any molecular tools to study how dismantling and re-assembly of 3D architecture is executed and coordinated with transcription. Our long-term goal is a complete understanding of the 3D choreography of replication over the course of S phase and its coordination with transcription. The overall objective of this application is to obtain direct measurements of replicon organization during S phase and model their 3D organization. Our central hypothesis is that replication initiation occurs stochastically at several (of many) potential origins that are in close 3D proximity at the time of initiation, after which forks remain in close proximity as chromatin transiently disengages from transcription and interphase 3D interactions. Our rationale is that high resolution single molecule 3D maps of nascent DNA will uncover novel mechanistic insights into how replication is faithfully executed and coordinated with transcription. AIM1 will develop a transformative single DNA fiber optical replication mapping (ORM) method, permitting us to map origins and fork polarities on single molecules with unprecedented throughput (30Gb/hr). We will integrate these maps with high resolution Repli-seq and Hi-C maps to reveal how replicons are organized in time and space. To model the native 3D structure of individual replisomes, we will develop replication fork-enriched versions of single-particle SPRITE (split pool recognition of interactions by tag extension), Hi-C and single cell Hi-C. SPRITE enables detection of multiple simultaneously occurring DNA and RNA interactions within cross- linked and individually bar-coded large chromatin complexes. In AIM2, we will capture complexes containing pulse-labeled nascent DNA (Repli-SPRITE) to assess 3D association of DNA and RNA, including nascent RNA, with active replication forks (i.e. replisomes). In AIM3, we will map DNA in close proximity to active replication forks by capturing pulse-labeled nascent DNA from Hi-C libraries (Repli-Hi-C). Population Repli-Hi-C will provide a high resolution global view of how contacts differ as replication forks pass through domains, while single cell Repli-Hi-C will enable 3D models of how multiple replicons are organized within domains and how replication is temporally coordinated across the genome in each cell. Importantly, AIMs 2 and 3 will also chase the labeled DNA before capture to track the dynamic re-assembly of interphase 3D structures. We expect to deliver an unprecedented view of how the human genome is organized for DNA replication and how replication is coordinated with 3D architecture and transcription. This contribution will be significant because it will deepen our understanding of how DNA replication is orchestrated to preserve genome integrity and cell-type specific chromatin architectures. The proposed research is innovative because it will disrupt paradigms in genome research and DNA replication, and open new horizons by developing methods to model 3D organization of any process involving DNA synthesis (e.g. replication, recombination, repair, chromatin assembly).